Figure 2
Effect of sphingomyelinase on chondrocyte function. (a) Sphingomyelinase treatment dose dependently induces cell death and decreases cell number. Chondrocytes were cultured for 24 hours in the presence of SMase (0–1.0 U/ml). Cell death and cell number were assessed using the CytoTox 96® assay (Promega), which quantitatively measures lactate dehydrogenase released into the media upon cell death during the culture period or upon lysis of living cells at the end of the culture period. Data shown are mean absorbance units (492 nm) ± standard error. ** P < 0.01 versus control. (b) Short-term SMase treatment induces proteoglycan release from articular chondrocytes. Chondrocytes were cultured for 24 hours in the presence of SMase (0–1.0 U/ml). Media was analyzed for release of sGAGs using the DMMB assay. Differences in release of sGAG associated with culture treatment are expressed as mean sGAG released per cell (mg/ml) ± standard error. * P < 0.05; ** P < 0.01. (c) SMase dose dependently increases cellular protein content. Following 24 hours of treatment with increasing doses of SMase, cells (with cell-associated matrix proteins) were solubilized with 0.9% Triton X-100 and the protein content (mg/ml) determined using the BCA assay (Pierce). Data are presented as mean ± standard error. * P < 0.05; *** P < 0.001, versus control. (d) Long-term SMase treatment reduces cell proliferation. Chondrocytes were cultured for 1–7 days and the effect of SMase (0.1 U/ml) on cell number determined using the CytoTox® assay. Data shown are mean absorbance units (492 nm) ± standard error. * P < 0.05 versus control at equivalent time point. (e) Long-term SMase treatment induces proteoglycan release from articular chondrocytes. Chondrocytes were cultured in the presence of SMase (0.1 U/ml) for 7 days. The amount of sGAG released into the media per cell was determined as above. Data are presented as mean ± standard error. * P < 0.05. DMMB, dimethylmethylene blue; sGAG = sulphated glycosaminoglycan; SMase, sphingomyelinase.